Re: [ccp4bb] Homology modeling

[Randy Read]

Dear Amit,

I don’t think that PROCHECK is the right tool for assessing the quality of a 
comparative model.  It was revolutionary in its time, but the criteria it looks 
at can mostly be satisfied by energy minimisation even of an incorrect model, 
especially if you don’t have to simultaneously satisfy experimental data.  For 
evaluating refined experimental models, I would now be inclined to use 
Molprobity (available from a website, CCP4 and Phenix) or tools built into 
graphics programs like ISOLDE and coot.

Model quality assessment is actually a thriving subfield in the protein 
modeling community and it has its own category in the CASP modeling challenges. 
 You want to be looking at the methods that have been judged best by seeing how 
well they perform in blind predictions.  Comparative models differ from 
experimental models in that they haven’t been constrained by the requirement to 
fit experimental data, so quality assessment has to look at more subtle 
features like residue environment.

The most recent CASP was CASP13, so a good start would be the main model 
quality assessment evaluation in the special issue from that event: 
https://onlinelibrary.wiley.com/doi/10.1002/prot.25767. You’ll find a variety 
of good tools described there.  The one we’ve been playing with, in terms of 
local assessment of model quality for MR, is ProQ3D, which has an online 
server: http://proq3.bioinfo.se.

Best wishes,
Randy Read

-
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research Tel: +44 1223 336500
The Keith Peters Building   Fax: +44 1223 336827
Hills Road   E-mail: 
rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.  
www-structmed.cimr.cam.ac.uk

> On 15 Aug 2020, at 00:39, amit gaur  wrote:
> 
> Hi All,
>I am trying to generate a model using comparative modeling. Can anybody 
> suggest the quality of the model based on procheck summary.
> 
>  +--<<<  P  R  O  C  H  E  C  K S  U  M  M  A  R  Y  
> >>>--+
>  |
> |
>  | /var/www/PROCHECK/Jobs/7358861/7358861.pdb   1.5 1253 residues 
> |
>  |
> |
> *| Ramachandran plot:   87.9% core   10.2% allow1.4% gener0.4% disall 
> |
>  |
> |
> *| All Ramachandrans:   67 labelled residues (out of1235) 
> |
> +| Chi1-chi2 plots:  3 labelled residues (out of 771) 
> |
>  | Side-chain params:5 better 0 inside  0 worse   
> |
>  |
> |
> *| Residue properties: Max.deviation: 4.9  Bad contacts:0 
> |
> *| Bond len/angle:9.9Morris et al class:  1  1  2 
> |
>  |
> |
>  | G-factors   Dihedrals:  -0.13  Covalent:  -0.07Overall:  -0.10 
> |
>  |
> |
> *| Planar groups:90.0% within limits  10.0% highlighted  13 off graph 
> |
>  |
> |
>  
> ++
>+ May be worth investigating further.  * Worth investigating further.
> 
> 
> 
> Thanks,
> 
> 
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[ccp4bb] Does MicroED (microcrystal electron diffraction) have phase problem ?

[Alex Lee]

Hi, All,

I am new to MicroED (microcrystal electron diffraction). I know that X-ray
crystallography has phase problem, and I think MicroED has phase problem
too (it is diffraction of electron instead of x-ray). However, when I read
the Wikipedia, I could not understand the following description of MicroED: One
of the main difficulties in X-ray crystallography is determining phases in
the diffraction pattern. Because of the complexity of X-ray lenses, it is
difficult to form an image of the crystal being diffracted, and hence phase
information is lost. Fortunately, electron microscopes can resolve atomic
structure in real space and the crystallographic structure factor phase
information can be experimentally determined from an image's Fourier
transform. The Fourier transform of an atomic resolution image is similar,
but different, to a diffraction pattern—with reciprocal lattice spots
reflecting the symmetry and spacing of a crystal.

Does the above description mean that MicroED, or more broadly electron
crystallogaphy does NOT suffer from phase problem?  How about single
particle cryo electron microscopy, it should NOT have phase problem, right?

Thanks for any input in it.

Best,
Alex



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Re: [ccp4bb] Does MicroED (microcrystal electron diffraction) have phase problem ?

[Jessica Bruhn]

Hi Alex,

Welcome to the field of microED! From a practical standpoint, microED also
suffers from the phase problem, and somewhat moreso compared to X-ray
crystallography because anomalous signal is very limited. It is true that a
TEM microscope operated in imaging mode produces images that contain both
phase and amplitude information, which you correctly infer means that
single particle cryoEM does not suffer from the phase problem. This is why
a map from cryoEM is generally of higher quality than one determined by
X-ray crystallography at the same resolution, because we don't have to
guess at the phases.

In classic microED data collection, we don't actually take advantage of the
phase information that can be measured using imaging mode. We quickly find
our crystals in imaging mode and then flip the switch to diffraction mode
and collect a dataset devoid of phase information. It has been suggested
that we could use imaging mode to get at the phases of the diffraction
patterns, but I am not aware of anyone actually doing this. Practically
speaking, this would add significant additional time to the data collection
and we likely would only be able to reliably use the phases for the lower
resolution range (though, that might not be a deal breaker). Thankfully
though, molecular replacement and ab initio methods for small molecules
work pretty well on these datasets. Plus, most X-ray structures are solved
this way anyways.

There have been efforts to use radiation damage as a means to phase a small
peptide bound to zinc (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7313391/).
And there is some interesting work being done with dynamical scattering as
a way to see differences in Friedel pairs (see Tim Gruene's post here:
https://www.jiscmail.ac.uk/cgi-bin/wa-jisc.exe?A2=CCP4BB;b22eae56.2007),
but these methods are not "classic microED" and require completely
different data processing software that is unfamiliar to most X-ray
crystallographers.

For now, we just crank up the cycles in SHELXT/SHELXD or hope for a good
molecular replacement model.

Best of luck,
Jessica

On Sat, Aug 15, 2020 at 6:03 PM Alex Lee  wrote:

> Hi, All,
>
> I am new to MicroED (microcrystal electron diffraction). I know that X-ray
> crystallography has phase problem, and I think MicroED has phase problem
> too (it is diffraction of electron instead of x-ray). However, when I read
> the Wikipedia, I could not understand the following description of MicroED: 
> One
> of the main difficulties in X-ray crystallography is determining phases in
> the diffraction pattern. Because of the complexity of X-ray lenses, it is
> difficult to form an image of the crystal being diffracted, and hence phase
> information is lost. Fortunately, electron microscopes can resolve atomic
> structure in real space and the crystallographic structure factor phase
> information can be experimentally determined from an image's Fourier
> transform. The Fourier transform of an atomic resolution image is similar,
> but different, to a diffraction pattern—with reciprocal lattice spots
> reflecting the symmetry and spacing of a crystal.
>
> Does the above description mean that MicroED, or more broadly electron
> crystallogaphy does NOT suffer from phase problem?  How about single
> particle cryo electron microscopy, it should NOT have phase problem, right?
>
> Thanks for any input in it.
>
> Best,
> Alex
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>


-- 
Jessica Bruhn, Ph.D
Principal Scientist
NanoImaging Services, Inc.
4940 Carroll Canyon Road, Suite 115
San Diego, CA 92121
Phone #: (888) 675-8261
www.nanoimagingservices.com



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[ccp4bb] Cell disruption

[Bernhard Lechtenberg]

Dear colleagues,

We are currently looking to purchase a cell disruptor/homogeniser mainly for 
routinely processing a few 100 mls of E. coli suspensions. With the current 
COVID-19 restrictions it is very difficult for us to test any equipment. I thus 
hope that some of you can share their experiences with the different models. I 
found a similar thread on the CCP4BB from 2013 but wondered if anybody had had 
some more up to date information.

We are mainly looking at the Avestin Emulsiflex C3 homogeniser and the 
Microfluidics LM20 Microfluidizer. In particular we are interested to know more 
about ease of use, maintenance, reliability and if anybody operates these in a 
cold room (4°C).

Thanks in advance,

Bernhard


--
Bernhard C. Lechtenberg, Ph.D.
Laboratory Head
Ubiquitin Signalling Division
The Walter and Eliza Hall Institute
1G Royal Parade
Parkville VIC 3052
Australia
Phone: +61 3 9345 2217
Email: lechtenber...@wehi.edu.au


___

The information in this email is confidential and intended solely for the 
addressee.
You must not disclose, forward, print or use it without the permission of the 
sender.

The Walter and Eliza Hall Institute acknowledges the Wurundjeri people of the 
Kulin
Nation as the traditional owners of the land where our campuses are located and
the continuing connection to country and community.
___



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Re: [ccp4bb] Cell disruption

[Pascal Egea]

Dear Bernhard ,
I would recommend the emulsified from Avestin. It is great . Depending on
your budget you can ask for stainless  steel (probe  to slow tear ) or
ceramic (longer lasting but a bit more expensive).
We have the ceramic but I have worked with the stainless steel one and it
is really good with E. coli cells. Not the best for
Yeast despite pressurizing more. Yeast are better dealt with bead beaters
or cryo-mill grinders.
3 passes are usually sufficient to process up to 250 ml of extract in a
decent time (15-20) minutes .

We operate ours at room temperature just cool the serpentine tubing coming
out of the chamber in ice . There is an option to add a cooling plate but
we have never considered it. Overheating comes from overpressurizing but
for E Coli at 15000 psi max  3 passes will do the trick without overhea tu
bf the sample. I have processEs successfully many membrane proteins And
protein complexes with it.

I have never seen one in a cold room to be honest but it should be fine
although I don’t think it would be necessary . I would Ask avestin about
that .
Maintenance and cleaning is simple . Once in a while I will replace the
seal, it s not very difficult to do. The whole assembly is metal so you can
sterilize the whole thing After complete disassembly in extreme cases .
This is a bit more involved but not overwhelming.
The people at Avestin are super nice and responsive ( Canadians) so they
will guide you if necessary . And we have had their engineers show up
sometimes to just check the instrument for free . I have had this one for
10 years in my lab now. And before that was using one for 7 years during my
post doc.  I would not lyse bacteria by any other method now.

I hope this helps.
All the best.
Pascal

On Sat, Aug 15, 2020 at 9:08 PM Bernhard Lechtenberg <
lechtenber...@wehi.edu.au> wrote:

> Dear colleagues,
>
>
>
> We are currently looking to purchase a cell disruptor/homogeniser mainly
> for routinely processing a few 100 mls of E. coli suspensions. With the
> current COVID-19 restrictions it is very difficult for us to test any
> equipment. I thus hope that some of you can share their experiences with
> the different models. I found a similar thread on the CCP4BB from 2013 but
> wondered if anybody had had some more up to date information.
>
>
>
> We are mainly looking at the Avestin Emulsiflex C3 homogeniser and the
> Microfluidics LM20 Microfluidizer. In particular we are interested to know
> more about ease of use, maintenance, reliability and if anybody operates
> these in a cold room (4°C).
>
>
>
> Thanks in advance,
>
>
>
> Bernhard
>
>
>
> 
>
>
>
> 
>
> --
>
> Bernhard C. Lechtenberg, Ph.D.
>
> Laboratory Head
>
> Ubiquitin Signalling Division
>
> The Walter and Eliza Hall Institute
>
> 1G Royal Parade
> 
>
> Parkville VIC 3052
> 
>
> Australia
> 
>
> Phone: +61 3 9345 2217
>
> Email: lechtenber...@wehi.edu.au
>
>
>
> ___
>
> The information in this email is confidential and intended solely for the
> addressee.
> You must not disclose, forward, print or use it without the permission of
> the sender.
>
> The Walter and Eliza Hall Institute acknowledges the Wurundjeri people of
> the Kulin
> Nation as the traditional owners of the land where our campuses are
> located and
> the continuing connection to country and community.
> ___
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>
-- 
Pascal F. Egea, PhD
Associate Project Scientist
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pegea at mednet.ucla.edu



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